The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein–protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall–targeting β-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein–protein interaction as one way of regulating its activity.

SummaryMany cases of active tuberculosis are thought to result from the reactivation of dormant Mycobacterium tuberculosis from a prior infection, yet remarkably little is known about the mechanism by which these non-sporulating bacteria reactivate. A family of extracellular bacterial proteins, known as resuscitation-promoting factors (Rpfs), has previously been shown to stimulate growth of dormant mycobacteria. While Rpf proteins are clearly peptidoglycan glycosidases, the mechanism and role of Rpf in mediating reactivation remains unclear. Here we use a yeast two-hybrid screen to identify potential binding partners of RpfB and report the interaction between RpfB and a putative mycobacterial endopeptidase, which we named Rpf-interacting protein A (RipA). This interaction was confirmed by in vitro and in vivo co-precipitation assays. The interacting domains map to the C-termini of both proteins, near predicted enzymatic domains. We show that RipA is a secreted, cell-associated protein, found in the same cellular compartment as RpfB. Both RipA and RpfB localize to the septa of actively growing bacteria by fluorescence microscopy. Finally, we demonstrate that RipA is capable of digesting cell wall material and is indeed a peptidoglycan hydrolase. The interaction between these two peptidoglycan hydrolases at the septum suggests a role for the complex in cell division, possibly during reactivation.

Polyprenylphosphate-arabinose (in which the polyprenyl unit is found both as decaprenyl and octahydroheptaprenyl) is a donor of mycobacterial cell wall arabinosyl residues. Because of this important role, its biosynthetic pathway, and that of the related lipid, polyprenylphosphate-D-ribose, was investigated. Further experiments showed that the mature polyprenylphosphate-ribose is formed from phosphoribose pyrophosphate via a two-step pathway involving a transferase to form polyprenylphosphate-5-phosphoribose and then a phosphatase to form the final polyprenylphosphateribose. Polyprenylphosphate-arabinose is formed by a similar pathway with an additional step being the epimerization at C-2 of the ribosyl residue. This epimerization occurs at either the level of phosphoribose pyrophosphate or at the level of polyprenylphosphate-5-phosphoribose.The mycobacterial cell wall core consists of a highly impermeable layer of the unique 70 -90 carbon mycolic acids covalently attached to an inner peptidoglycan layer by way of the connecting polysaccharide, arabinogalactan. Arabinogalactan consists of three regions: the linker region (1), which is connected to the peptidoglycan, a galactan directly attached to the linker (2), and an arabinan (2), which is directly attached to the galactan. The mycolic acids are attached at the non-reducing end (3) of the arabinan. Since the arabinan is fundamental to the structural integrity of the cell wall, its biosynthesis has recently been studied (4 -9) with an ultimate aim of developing new tuberculosis drugs targeted at one or more of the arabinose biosynthetic enzymes.A major breakthrough (4) in these studies was the isolation of polyprenylphosphate-arabinose in the form of decaprenylphosphate-arabinose and octahydroheptaprenylphosphatearabinose. Subsequently, it was demonstrated that radioactive decaprenylphosphate-arabinose made chemically (5), or a mixture of radioactive decaprenylphosphate-arabinose and octahydroheptaprenylphosphate-arabinose, isolated from cultures of Mycobacterium smegmatis, 1 could function as arabinosyl donors in the presence of M. smegmatis enzymes to form polymeric arabinan. Hence, determining the pathway of the biosynthesis of polyprenylphosphate-arabinose itself becomes important for the ultimate goal of developing new drugs against mycobacteria.In a related area, mycobacteria have been shown (9) to synthesize polyprenylphosphate-ribose. This compound has been characterized as decaprenylphosphate-ribose (9), although it is likely to exist as octahydroheptaprenylphosphate-ribose as well. The function of this glycolipid is not yet clear. It does not appear to be a biosynthetic precursor of polyprenylphosphatearabinose because radioactive polyprenylphosphate-ribose is not converted by mycobacterial enzymes to polyprenylphosphate-arabinose (9). Polyprenylphosphate-ribose may function as a ribosyl donor (9) since mycobacteria have been shown to ribosylate certain antibiotics (10).Polyprenylphosphate sugars are generally synthesized by the transfer of a glycosy...

Ethambutol is known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan (K. Takayama and J. O. Kilburn, Antimicrob. Agents Chemother. 33:1493-1499, 1989). This effect was confirmed, and it was also shown that ethambutol inhibits biosynthesis of the arabinan of lipoarabinomannan, a lipopolysaccharide noncovalently associated with the cell wall core. In contrast to cell wall core arabinan, which is completely inhibited by ethambutol, synthesis of the arabinan of lipoarabinomannan was only partially affected, demonstrating a differential effect on arabinan synthesis in the two locales. Further studies of the effect of ethambutol on cell wall biosynthesis revealed that the synthesis of galactan in the cell wall core is strongly inhibited by the drug. In addition, ethambutol treatment resulted in the cleavage of arabinosyl residues present in the mycobacterial cell wall; more than 50% of the arabinan in the cell wall core was removed from the wall 1 h after addition of the drug to growing mycobacterial cultures. In contrast, galactan was not released from the cell wall during ethambutol treatment. The natural function of the arabinosyl-releasing enzyme remains unknown, but its action in combination with inhibition of synthesis during ethambutol treatment results in severe disruption of the mycobacterial cell wall. Accordingly, ethambutol-induced damage to the cell wall provides a ready molecular explanation for the known synergetic effects of ethambutol with other chemotherapeutic agents. Nevertheless, the initial direct effect of ethambutol remains to be elucidated.

The capsular polysaccharide of group B Streptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with ␤-Nacetylglucosaminidase, which catalyzes hydrolysis of the ␤-D-GlcNAc(134)␤-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and 31 P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.